Analysis of Nonylphenol:  Advances and Improvements in the Immunochemical Determination Using Antibodies Raised against the Technical Mixture and Hydrophilic Immunoreagents

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of technical nonylphenol (NP) is reported. The preparation of specific antibodies has been addressed using an immunizing hapten with a four-carbon atom spacer arm placed at the ortho position that preserves both the hydroxyl group and the complexity of the branched nonyl chain mixture of the technical NP. The synthesis of the immunizing hapten 5-(2-hydroxy-5-nonylphenyl)-pentanoic acid has been accomplished through a four-step synthetic pathway using the NP commercial technical mixture as the starting material. Three types of competitor haptens have also been prepared depending on the location of the spacer arm:  in ortho position to the phenol group (type A), attached to the oxygen atom (type B), and in para position, substituting the nonyl chain (type C). Drawbacks produced by the hydrophobicity of the NP or of the hapten derivatives have been circumvented by using a highly hydrophilic carrier molecule such as a high-molecular-weight aminodextran as a coating support for antigen in an indirect ELISA format. A reproducible and sensitive indirect competitive ELISA has been finally obtained, reaching a limit of detection of 2.3 ± 0.9 μg L^-1 and an IC₅₀ value of 29 ± 5 μg L^-1 (both N = 16). A coefficient of variation of 11% for assays performed on different days (N = 5; IC₅₀ = 30 ± 3 μg L^-1) demonstrates the assay reproducibility. The assay also recognizes the nonylphenol polyethoxylates to a different degree depending on the length of the ethoxylate chain. Recovery values in the range between 96 and 100% have been obtained using spiked blind aqueous samples although the sample preparation procedure used has been shown to have a great influence on the method accuracy. A preliminary evaluation of the analytical protocol established has been performed using real water samples.